Subcellular fractionation of rat liver homogenates using two-polymer phase systems in a toroidal-coil centrifuge.

The principal organelles of rat liver homogenates were fractionated by two-phase partition chromatography using toroidal-coil centrifugation with a mixture of dextran T 500 and poly(ethylene glycol) 6000 in 0.26 M-sucrose containing 10 mM-sodium phosphate/phosphoric acid buffer, pH 7.4. The effects of varying the following parameters on organelle elution profiles, as reflected by their marker-enzyme activities, were studied: centrifuge speed; the composition and relative proportion of dextran-rich and poly(ethylene glycol)-rich phases in the eluent; flow rate; sample volume; homogenate concentration; helix diameter; tubing bore and the number of loops in the coil. Optimal resolution of the organelles was achieved with a toroidal coil of internal diameter 1.07 mm with a 4.55 mm helix diameter on a 0.42 m-diameter rotor running at 1000 rev./min. The eluent was prepared by combining, in a ratio of 93:7 (v/v), the poly(ethylene glycol)-rich upper phase and dextran-rich lower phase obtained from a phase mixture containing 3.3% (w/w) dextran and 5.4% (w/w) poly(ethylene glycol). The flow rate of the eluent was 14ml/h. Optimal conditions for separation of the organelles were evaluated. Resolution of plasma membrane and lysosomes was achieved. Separation of endoplasmic reticulum, which showed marked heterogeneity, from plasma membrane was also demonstrated. DNA and marker enzymes for peroxisomes, mitochondria and cytosol showed distinct elution profiles.     

The partition of glycosaminoglycan-quaternary ammonium complexes. I. The effect of phase composition.

The complexes formed between quaternary ammonium cations and polyanionic glycosaminoglycans can be partitioned between partially miscible aqueous inorganic salt and alcohol phases. Small changes in salt concentration can completely shift the complex from one phase to the other. The effect of the phase composition variables: the type of inorganic salt, the type of quaternary ammonium salt, and the alcohol used, were systematically investigated. The sharp transition from solubility in the upper non-aqueous phase to solubility in the lower, aqueous phase was found to be strongly affected by the type of inorganic salt. This transition occurred at higher salt concentrations when NaCl, KCl, or LiCl were used than when CaCl2 or MgCl2 were used. Differences in behavior among glycosaminoglycans were larger for NaCl than for CaCl2. The complex is stabilized to dissociation by salt by increasing hydrophobicity of the non-aqueous phase. However, aggregation of the complex into an insoluble form is also favored by an increasingly hydrophobic environment. The most consistent partition was observed with 1- and 2-butanol. The partition isotherm of chondroitin 4-sulfate was investigated at constant salt concentration. It was found that the partition coefficient varies with the concentration of chondroitin 4-sulfate, although the magnitude of this effect could be diminished by increasing the quaternary ammonium salt concentration.     

The partition of glycosaminoglycan-quaternary ammonium complexes: I. The effect of phase composition

The complexes formed between quaterny ammonium cations and polyanionic glycosaminoglycans can be partitioned between partially miscible aqueous inorganic salt and alcohol phases. Small changes in salt concentration can completely shift the complex from one phase to the other. The effect of the phase composition variables: the type of inorganic salt, the type of quaternary ammonium salt, and the alcohol used, were systematically investigated. The sharp transition from solubility in the upper non-aqueous phase to solubility in the lower, aqueous phase was found to be strongly affected by the type of inorganic salt. This transition occurred at higher salt concentrations when NaCl, KCl, or LiCl were used than when CaCl₂ or MgCl₂ were used. Differences in behavior among glycosaminoglycans were larger for NaCl than for CaCl₂. The complex is stabilized to dissociation by salt by increasing hydrophobicity of the non-aqueous phase. However, aggregation of the complex into an insoluble form is also favored by an increasingly hydrophobic environment. The most consistent partition was observed with 1- and 2-butanol. The partition isotherm of chondroitin 4-sulfate was investigated at constant salt concentration. It was found that the partition coefficient varies with the concentration of chondroitin 4-sulfate, although the magnitude of this effect could be diminished by increasing the quaternary ammonium salt concentration.     

Organelle association visualized by three-dimensional ultrastructural imaging of the yeast cell

This study was aimed at a better understanding of organelle organization in the yeast Saccharomyces cerevisiae with special emphasis on the interaction and physical association of organelles. For this purpose, a computer aided method was employed to generate three-dimensional ultrastructural reconstructions of chemically and cryofixed yeast cells. This approach showed at a high level of resolution that yeast cells were densely packed with organelles that had a strong tendency to associate at a distance of <30 nm. The methods employed here also allowed us to measure the total surface area and volume of organelles, the number of associations between organelles, and the ratio of associations between organelles per surface area. In general, the degree of organelle associations was found to be much higher in chemically fixed cells than in cryofixed cells, with endoplasmic reticulum/plasma membrane, endoplasmic reticulum/mitochondria and lipid particles/nuclei being the most prominent pairs of associated fractions. In cryofixed cells, similar preferences for organelle association were seen, although at lower frequency. The occurrence of specific organelle associations is believed to be important for intracellular translocation and communication. Membrane contact as a possible means of interorganelle transport of cellular components, especially of lipids, is discussed.     

Structural phase relationships in lecthin-cholate-water systems

In the first part of this paperthe anomalies and discontinuities observed in different physical properties of the lecithin-cholate isotropic phase (light scattering, sodium ion activity, viscosity, conductivity) as well as the results of an enzymatic study are explained in terms of partial aggregation which sets in when the intermicellar solution is “unsaturated” in cholate.It is proposed that this aggregation process in the isotropic phase is exactly the inverse process of the disintegration of the hexagonal phase when the latter is diluted. The mixed micelles had previously been shown to be in the form of a bimolecular discs, both the cylindrical elements of the hexagonal phase and the entities in the aggregation process are stacks of mixed micellar discs, the existence of both of these states being governed by the same delicate hydrophilic-hydrophobic balance.The arguments in favour of this are discussed in the second part of the paper where in particular the model proposed for the hexagonal phase is verified by a detailed calculation from X-ray diffraction measurements. The variations of both the water layer between discs in the cylinder and of the intercylinder water volume were shown to be coherent with changes in lecithin concentration and the amount of water present, in agreement with the above mentioned balance.Finally having demonstrated the structural resemblance between these two phases and the mechanism of the passage from one phase to the other, an explanation of the inter-relationship of all phases in the system is attempted.     

Regulation of the distribution of carotenoid droplets in goldfish xanthophores and possible implication to secretory processes

In goldfish xanthophores, the formation of pigment aggregate requires: 1) that a pigment organelle (carotenoid droplet) protein p57 be in the unphosphorylated state; 2) that self-association of pigment organelles occur in a microtubule-independent manner; and 3) that pigment organelles via p57 associate with microtubules. In the fully aggregated state, the pigment organelles are completely stationary. Pigment dispersion is initiated by activation of a cAMP-dependent protein kinase, which phosphorylates p57 and allows pigment dispersion via an active process dependent on F-actin and a cytosolic factor. This factor is not an ATPase, and its function is unknown. However, its abundance in different tissues parallels secretory activity of the tissues, suggesting a similarity between secretion and pigment dispersion in xanthophores. The identity of the motor for pigment dispersion is unclear. Experimental results show that pigment organelles isolated from cells with dispersed pigment have associated actin and ATPase activity comparable to myosin ATPase. This ATPase is probably an organelle protein of relative molecular mass approximately 72,000, and unlikely to be an ion pump. Isolated pigment organelles without associated actin have 5x lower ATPase activity. Whether this organelle ATPase is the motor for pigment dispersion is under investigation. The process of pigment aggregation is poorly understood, with conflicting results for and against the involvement of intermediate filaments.     

Cell surface energy, contact angles and phase partition. I. Lymphocytic cell lines in biphasic aqueous mixtures

The surface energy of cells is the quantity which dominates certain physical interactions of cells such as adhesion to hydrophobic surfaces and phagocytosis. A linear relationship is derived relating the equilibrium constant obtained from phase partition in liquid-liquid systems with the surface energy difference obtained from contact-angle measurements. Using biphasic mixtures of Dextran and poly(ethylene glycol) in a medium of constant salt composition the expression is confirmed for transformed lymphocytic cell lines. The results demonstrate the importance of van der Waals' interactions in the phase-partition process, that phase partition can be used as a direct measure of cell surface hydrophobicity, and that the equilibrium constant of phase partition is directly related to the difference in the surface energy of the partitioned particle between the two phases.     

Electrostatic modulation of hnRNPA1 low‐complexity domain liquid–liquid phase separation and aggregation

Membrane‐less organelles and RNP granules are enriched in RNA and RNA‐binding proteins containing disordered regions. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a key regulating protein in RNA metabolism, localizes to cytoplasmic RNP granules including stress granules. Dysfunctional nuclear‐cytoplasmic transport and dynamic phase separation of hnRNPA1 leads to abnormal amyloid aggregation and neurodegeneration. The intrinsically disordered C‐terminal domain (CTD) of hnRNPA1 mediates both dynamic liquid–liquid phase separation (LLPS) and aggregation. While cellular phase separation drives the formation of membrane‐less organelles, aggregation within phase‐separated compartments has been linked to neurodegenerative diseases. To understand some of the underlying mechanisms behind protein phase separation and LLPS‐mediated aggregation, we studied LLPS of hnRNPA1 CTD in conditions that probe protein electrostatics, modulated specifically by varying pH conditions, and protein, salt and RNA concentrations. In the conditions investigated, we observed LLPS to be favored in acidic conditions, and by high protein, salt and RNA concentrations. We also observed that conditions that favor LLPS also enhance protein aggregation and fibrillation, which suggests an aggregation pathway that is LLPS‐mediated. The results reported here also suggest that LLPS can play a direct role in facilitating protein aggregation, and that changes in cellular environment that affect protein electrostatics can contribute to the pathological aggregation exhibited in neurodegeneration.     

Solubility of amphiphiles in membranes: influence of phase properties and amphiphile head group

The solubilities of two fluorescent lipid amphiphiles with comparable apolar structures and different polar head groups, NBD-hexadecylamine and RG-tetradecylamine (or -octadecylamine), were compared in lipid bilayers at a molar ratio of 1/50 at 23 degrees C. Bilayers examined were in the solid, liquid-disordered, or liquid-ordered phases. While NBD-hexadecylamine was soluble in all the examined bilayer membrane phases, RG-tetradecylamine was stably soluble only in the liquid-disordered phase. RG-tetradecylamine insolubility in solid and liquid-ordered phases manifests itself as an aggregation of the amphiphile over a period of several days and the kinetics of aggregation were studied. Solubility of these amphiphiles in the different phases examined seems to be related to the dipole moment of the amphiphile (in particular, of the polar fluorophore) and its orientation relative to the dipolar potential of the membrane. We propose that amphiphilic molecules inserted into membranes (including lipid-attached proteins) partition into different coexisting membrane phases based upon: (1) nature of the apolar structure (chain length, degree of saturation, and chain branching as has been proposed in the literature); (2) magnitude and orientation of the dipole moment of the polar portion of the molecules relative to the membrane dipolar potential; and (3) hydration forces that are a consequence of ordering of water dipoles at the membrane surface.     

Getting RNA and protein in phase

Nonmembrane-bound organelles such as RNA granules behave like dynamic droplets, but the molecular details of their assembly are poorly understood. Several recent papers identify structural features that drive granule assembly, shedding light on how phase transitions functionally organize the cell and may lead to pathological protein aggregation.     

Evaluation of liquid chromatography column retentivity using macromolecular probes. IV. Poly(ethylene glycol) bonded phase

Interaction properties of the novel HPLC silica gel-poly(ethylene glycol) (PEG) bonded phase were evaluated applying polymeric test substances, viz. polystyrenes, poly(methyl methacrylate)s, poly(ethylene oxide)s and poly(2-vinyl pyridine)s, and eluents of different polarities. Silanols on the silica gel surface are well shielded by the PEG phase, and silanophilic adsorption of macromolecules is suppressed in comparison with most silica C(18) bonded phases. The adsorption of solutes on the -OH groups of the PEG phase seems to be low as well. The partition of macromolecules in favor of the PEG phase is inferior to that observed in case of the silica C(18) phases. The volume of the PEG bonded phase is small and it is supposed that the PEG chains assume flat conformation on the silica gel surface.     

Concerning the separation of mammalian cells in immobilized metal ion affinity partitioning systems: a matter of selectivity

The effect of introducing an immobilized metal ion ligand in the lower phase of the PEG/Dextran system was studied on the erythrocytes and lymphocytes partition. The ligand in the lower phase was added as an insoluble form [Sepharose-IDA-M(II)] with or without a ligand in the upper phase. We first checked that the addition of the insoluble ligand in the system did not affect the phase volume and settling, and also that Sepharose-IDA-M(II) partitioned strictly in the lower phase. Then we studied the partition of cells with various concentrations of ligand in the lower and upper phases. We clearly demonstrate here that the partition in immobilized metal ion affinity partitioning (IMAP) systems is correlated with the affinity between the cell surface and the ligand. Cells are attracted to the ligand-containing phase. This fact is important not only for the greater understanding of IMAP, but could also for the separation of some types of cells. © 1998 John Wiley & Sons, Ltd.     

On the use of partition coefficients to characterize the distribution of fluorescent membrane probes between coexisting gel and fluid lipid phase: an analysis of the partition behavior of 1,6-diphenyl-1,3,5-hexatriene

The distribution of the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene between coexisting gel and fluid phospholipid phases in multilamellar vesicles has been examined using fluorescence quenching by spin-labeled phosphatidylcholine. For both thermally-induced and Ca2+-induced lipid phase separation, the ratio of probe concentration in the fluid liquid-crystal phase to that in the gel phase is found to be independent of either the probe concentration or the relative amounts of gel and fluid lipid phases, and hence is an equilibrium concentration ratio, or partition coefficient.     

Cell cycle maintenance and biogenesis of the Golgi complex

How organelle identity is established and maintained, and how organelles divide and partition between daughter cells, are central questions of organelle biology. For the membrane-bound organelles of the secretory and endocytic pathways [including the endoplasmic reticulum (ER), Golgi complex, lysosomes, and endosomes], answering these questions has proved difficult because these organelles undergo continuous exchange of material. As a result, many "resident" proteins are not localized to a single site, organelle boundaries overlap, and when interorganellar membrane flow is interrupted, organelle structure is altered. The existence and identity of these organelles, therefore, appears to be a product of the dynamic processes of membrane trafficking and sorting. This is particularly true for the Golgi complex, which resides and functions at the crossroads of the secretory pathway. The Golgi receives newly synthesized proteins from the ER, covalently modifies them, and then distributes them to various final destinations within the cell. In addition, the Golgi recycles selected components back to the ER. These activities result from the Golgi's distinctive membranes, which are organized as polarized stacks (cis to trans) of flattened cisternae surrounded by tubules and vesicles. Golgi membranes are highly dynamic despite their characteristic organization and morphology, undergoing rapid disassembly and reassembly during mitosis and in response to perturbations in membrane trafficking pathways. How Golgi membranes fragment and disperse under these conditions is only beginning to be clarified, but is central to understanding the mechanism(s) underlying Golgi identity and biogenesis. Recent work, discussed in this review, suggests that membrane recycling pathways operating between the Golgi and ER play an indispensable role in Golgi maintenance and biogenesis, with the Golgi dispersing and reforming through the intermediary of the ER both in mitosis and in interphase when membrane cycling pathways are disrupted.     

Dynamic instability and motile events of native microtubules from squid axoplasm

Native microtubules from extruded axoplasm of squid giant axons were used as a paradigm to characterize the motion of organelles along free microtubules and to study the dynamics of microtubule length changes. The motion of large round organelles was visualized by AVEC-DIC microscopy and analyzed at a temporal resolution of 10 frames per second. The movements were smooth and showed no major changes in velocity or direction. During translocation, the organelles paused very rarely. Superimposed on the rather constant mean velocity was a velocity fluctuation, which indicated that the organelles are subject to considerable thermal motion during translocation. Evidence for a regular low-frequency oscillation was not found. The thermal motion was anisotropic such that axial motion was less restricted than lateral motion. We conclude that the crossbridge connecting the moving organelle to the microtubule has a flexible region that behaves like a hinge, which permits preferential movement in the direction parallel to the microtubule. The dynamic changes in length of native microtubules were studied at a temporal resolution of 1 Hz. About 98% of the native microtubules maintained their length ("stable" microtubules), while 2% showed phases of growing and/or shrinking typical for dynamic instability ("dynamic" microtubules). Gliding and organelle motion were not influenced by dynamic length changes. Transitions between growing and shrinking phases were low-frequency events (1-10 minutes per cycle). However, a new type of microtubule length fluctuation, which occurred at a high frequency (a few seconds per cycle), was detected. The length changes were in the 1-3 micron range. The latter events were very prominent at the (+) ends. It appears that the native axonal microtubules are much more stable than the purified microtubules and the microtubules of cultured cells that have been studied thus far. Potential mechanisms accounting for the three states of microtubule stability are discussed. These studies show that the native microtubules from squid giant axons are a very useful paradigm for studying microtubule-related motility events and microtubule dynamics.     

A quantitative dynamic concept of the interphase partition of lipids: application to bile salt-lecithin-cholesterol mixed micelles

A system is proposed for a quantitative classification of lipids, based on interphase partition coefficients. This system enables calculation of exchanges of lipid molecules between phases. The mass/volume chemical unit mol X cm-3, strictly derived from the CGS system, is used, thus simplifying mathematical relations. Applied to bile salt-lecithin-cholesterol mixed micelles, this dynamic concept gives new insight into the variations of physico-chemical parameters. Experimental results obtained with the glycodesoxycholate and the taurocholate show a striking difference in partition coefficients between aqueous and mixed bile salt-lecithin interfacial phases. A new model applying triangular co-ordinates to a bile salt-lecithin-cholesterol mixed lipid phase is described.     

Cytoplasmic reorganization during the resumption of meiosis in cultured preovulatory rat oocytes

Changes in organelle topography and microtubule configuration have been studied during the resumption and progression of meiosis in cultured preovulatory rat oocytes. Germinal vesicle breakdown (GVBD) was reversibly inhibited by dibutyryl cAMP (DcAMP) or nocodazole, a microtubule-disrupting agent. The microtubule stabilizing agent taxol did not inhibit GVBD, but did impair further maturation. The migration of acidic organelles and chromatin in living oocytes was analyzed using the vital stains acridine orange and Hoechst 33258, respectively. Germinal vesicle stage oocytes undergo perinuclear aggregation of acidic organelles during GVBD and these organelles subsequently disperse into the cell cortex as the first meiotic spindle migrates to the oocyte periphery. DcAMP and nocodazole block the perinuclear aggregation of acidic organelles, whereas, in taxol-treated oocytes, organelle aggregation and GVBD occur but the dispersion of acidic organelles was arrested. Dose-response studies on the effects of nocodazole showed that GVBD was generally retarded and that a 50% inhibition of GVBD was achieved at concentrations in excess of 1.0 microM. Concentrations of taxol at 10 microM or above effectively inhibited both chromatin condensation and meiotic spindle formation. Indirect immunofluorescence microscopy with anti-tubulin antibodies revealed dissolution of microtubules with 1.0 microM nocodazole. Taxol had little effect on microtubule organization in germinal vesicle or chromatin condensation stage oocytes; however, when oocytes that had formed first meiotic spindles were treated with taxol, numerous microtubule asters appeared which were preferentially associated with the oocyte cortex. The changes in organelle topography, microtubule configuration, and drug sensitivity are discussed with respect to the regulation of cytoplasmic reorganization during the meiotic maturation of rat preovulatory oocytes.     

When size does matter: organelle size influences the properties of transport mediated by molecular motors

Background

Organelle transport is driven by the action of molecular motors. In this work, we studied the dynamics of organelles of different sizes with the aim of understanding the complex relation between organelle motion and microenvironment.

Methods

We used single particle tracking to obtain trajectories of melanosomes (pigmented organelles in Xenopus laevis melanophores). In response to certain hormones, melanosomes disperse in the cytoplasm or aggregate in the perinuclear region by the combined action of microtubule and actin motors.

Results and conclusions

Melanosome trajectories followed an anomalous diffusion model in which the anomalous diffusion exponent (α) provided information regarding the trajectories' topography and thus of the processes causing it. During aggregation, the directionality of big organelles was higher than that of small organelles and did not depend on the presence of either actin or intermediate filaments (IF). Depolymerization of IF significantly reduced α values of small organelles during aggregation but slightly affect their directionality during dispersion.

General significance

Our results could be interpreted considering that the number of copies of active motors increases with organelle size. Transport of big organelles was not influenced by actin or IF during aggregation showing that these organelles are moved processively by the collective action of dynein motors. Also, we found that intermediate filaments enhance the directionality of small organelles suggesting that this network keeps organelles close to the tracks allowing their efficient reattachment. The higher directionality of small organelles during dispersion could be explained considering the better performance of kinesin-2 vs. dynein at the single molecule level.     

The phase behavior of mixed aqueous dispersions of dipalmitoyl derivatives of phosphatidylcholine and diacylglycerol.

The phases and transition sequences for aqueous dispersions of mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycerol (1,2-DPG) have been studied by differential scanning calorimetry, dynamic x-ray diffraction, freeze-fracture electron microscopy, 31P-nuclear magnetic resonance spectroscopy, and Fourier-transform infrared spectroscopy. The results have been used to construct a dynamic phase diagram of the binary mixture as a function of temperature over the range 20 degrees-90 degrees C. It is concluded that DPPC and 1,2-DPG form two complexes in the gel phase, the first one with a DPPC/1,2-DPG molar ratio of 55:45 and the second one at a molar ratio of approximately 1:2, defining three different regions in the phase diagram. Two eutectic points are postulated to occur: one at a very low 1,2-DPG concentration and the other at a 1,2-DPG concentration slightly higher than 66 mol%. At temperatures higher than the transition temperature, lamellar phases were predominant at low 1,2-DPG concentrations, but nonlamellar phases were found to be predominant at high proportions of 1,2-DPG. A very important aspect of these DPPC/1,2-DPG mixtures was that, in the gel phase, they showed a ripple structure, as seen by freeze-fracture electron microscopy and consistent with the high lamellar repeat spacings seen by x-ray diffraction. Ripple phase characteristics were also found in the fluid lamellar phases occurring at concentrations up to 35.6 mol% of 1,2-DPG. Evidence was obtained by Fourier transform infrared spectroscopy of the dehydration of the lipid-water interface induced by the presence of 1,2-DPG. The biological significance of the presence of diacylglycerol in membrane lipid domains is discussed.     

Kinetic model for membrane transport. 2. Time lag and overshoot

A lag time during the period of variation in solute concentration in the receiver phase and overshoot in that in the membrane phase have been predicted to occur with a kinetic model for membrane transport which takes into account both the membrane volume and the partitioning kinetics (Makino et al., Biophys. Chem. 35 (1990) 85). The duration of the lag time becomes longest when the donor and receiver phases have the same volume. This maximum grows in length with increase in the partition coefficient, tending to be proportional to the volume fraction of the receiver phase. Moreover, it displays an increase in length with decreasing membrane volume fraction. Overshoot occurs only when the volume fraction of the receiver phase is greater than that of the donor. Overshoot is observed during the earlier stages of membrane transport when the partition coefficient is smaller or the volume fraction of the receiver phase is larger.